Necrosis- and ethylene-inducing 1-like proteins (NLPs) constitute a superfamily of proteins found in diverse phyla of plant-associated microorganisms, such as bacteria, oomycetes, and fungi. So far, more than 1,700 homologs of these apoplastic effectors have been identified. Numerous NLPs are cytotoxic and help to facilitate infections towards a wide range of crops. The target for NLP interaction with the plant plasma membrane is glycosyl inositol phosphoramide (GIPC), a major sphingolipid found almost exclusively in plant plasma membrane. Furthermore, NLPs are toxic towards eudicot, but not against monocot plants. The structural comparison of eudicot and monocot GIPCs elucidates the difference in terminal sugar moieties, providing a possible explanation for the lack of toxicity towards monocots. Only three NLP 3D structures have been determined so far and they reveal NLPs have a β-sandwich fold similar to actinoporins, pore-forming toxins from sea anemones. In addition, studies of the structure and binding of NLPPya revealed important residues for interaction with the terminal hexose unit of GIPCs. However, it is unknown the GIPC-binding and mechanism of membrane damage, which is still largely unexplored, is universal for all toxic NLPs.
Numerous genera of bacteria (e.g. Pectobacterium), oomycetes (e.g. Phytophthora) and fungi (e.g. Moniliphtora) with diverse lifestyles secret NLPs and the exact role of NLPs in the infection cycle remains enigmatic. Structural and functional characterization of NLPs from evolutionary distant organisms is essential to understand the evolution and mechanistic action of these proteins. We expressed and studied two NLPs, NPPPp from Phytophthora parasitica and MpNEP2 from Moniliphtora perniciosa. Preliminary results, obtained by performing infiltration assays and conductivity measurements in Nicotiana tabacum, and combined with binding analysis by surface plasmon resonance and sedimentation assay utilizing multilamellar vesicles indicate that diverse NLPs share similar functional traits.